Intravenous (i.v.) injection of soluble antigens can induce antigen-specific tolerance in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, and effectively suppresses the disease. I.V. injected autoantigen induces tolerogenic dendritic cells (DCs), which produce IL-27, a potent immunoregulatory cytokine. While tolerance can be successfully induced in wild-type EAE mice, mice lacking IL-27 receptor are resistant, demonstrating that IL-27 plays a crucial role in tolerance induction. The central hypothesis of this proposal is that i.v. administration of autoantigen in EAE causes DCs to produce IL-27, which in turn induces tolerogenic DCs and regulatory Tr1 cells, leading to tolerance. To test this hypothesis, three specific aims are proposed: Aim 1) To study the role of IL-27 in generation of tolerogenic DCs and antigen-specific type 1 regulatory T (Tr1) cells in i.v. tolerance. We hypothesize that IL-27 plays a key role in i.v. tolerance by both paracrine (suppression of myelin-specific Th1/Th17 cells and induction of Tr1 cells) and autocrine (induction and expansion of tolerogenic DCs) mechanisms. Aim 2) To investigate the role of the galectin-1/IL-27 pathway in i.v. tolerance induction. Galectin-1 is an endogenous glycan-binding protein, produced by Tregs that can endow DCs with IL-27-dependent tolerogenic potential. We have found that MOG-reactive Tregs stimulate DCs to produce IL-27, an effect that is largely galectin-1-dependent. IL-27, in turn, can stimulate DCs to express a high level of galectin-1. Further, galectin-1 deficient mice are resistant to the induction of i.v. tolerance in EAE. In this aim we will test our hypothesis that i.v. injection of autoAg will trigger Ag-specific nTregs to preferentially form aggregates with DCs, produce galectin-1, and induce tolerogenic DCs in vivo. Aim 3) To test the effect of exogenous IL-27 on i.v. tolerance induction in EAE. Given the important role of IL- 27 in tolerance induction, we hypothesize that administration of rmIL-27 will promote development of tolerogenic/regulatory DCs and T cells upon i.v. injection of autoantigen. Hence, administration of exogenous IL-27 can enhance the therapeutic effect of i.v. tolerance, and reduce the dose of autoantigen required for maximal effect. This will decrease the likelihood of developing autoantibodies or adverse immune responses to the tolerizing autoantigen. Aims 1 and 2 study basic mechanisms of i.v. tolerance induction, while Aim 3 focuses on translation of our findings into therapy. The information gained from these studies should lead to a better understanding of the mechanisms of i.v. tolerance and provide the basis for novel immunotherapies for CNS inflammatory demyelination.